A self-complementary AAV proviral plasmid that reduces cross-packaging and ITR promoter activity in AAV vector preparations

Adeno-associated viral vectors (AAVs) are a leading delivery system for gene therapy in animal models and humans. With several Food and Drug Administration-approved AAV gene therapies on the market, issues related to vector manufacturing have become increasingly important. In this study, we focused on potentially toxic DNA contaminants that can arise from AAV proviral plasmids, the raw materials required for manufacturing recombinant AAV in eukaryotic cells. Typical AAV proviral plasmids are circular DNAs containing a therapeutic gene cassette flanked by natural AAV inverted terminal repeat (ITR) sequences, and a plasmid backbone carrying prokaryotic sequences required for plasmid replication and selection in bacteria. While the majority of AAV particles package the intended therapeutic payload, some capsids instead package the bacterial sequences located on the proviral plasmid backbone. Since ITR sequences also have promoter activity, potentially toxic bacterial open reading frames can be produced in vivo, thereby representing a safety risk. In this study, we describe a new AAV proviral plasmid for vector manufacturing that (1) significantly decreases cross-packaged bacterial sequences, (2) increases correctly packaged AAV payloads, and (3) blunts ITR-driven transcription of cross-packaged material to avoid expressing potentially toxic bacterial sequences. This system may help improve the safety of AAV vector products.

sequencing from AAV preps generated with the indicated proviral plasmids.Reads were mapped to the U6.mi405 insert and proviral backbone sequences, as well as "other" packaged DNA contaminants arising from the Ad pHELPER plasmid, pRep2/Cap"X" plasmids (where X = indicated serotype), or HEK293 cell host DNA.
Table S4.Top 10 reads from host DNA sequences packaged in AAV9 vectors Reads were identified from long-read sequencing of AAV9 vectors prepped with C1 or C5 proviral plasmid backbones.Packaged reads not mapping to insert, backbone, pHELPER, or Rep/Cap sequences were mapped to the human reference genome to characterize "host" cell DNA sequences and tabulated based on the genes (including introns) that they overlapped with by at least 15bp.The top hit in both preps was COL2A1, which is derived from the collagen intron stuffer sequence in our U6.mi405payload, but the other sequences appear to be randomly inserted into AAV genomes.
Table S5.Identification of truncations and truncation hot-spots in the U6.mi405 inserts produced using C1 or C5 backbones.Relevant features are indicated.The C5 insert is 180 nucleotides larger than the C1 insert due to the addition of two CTCF binding sites and two 3X stop codons.

Fig S1 .
Fig S1.Assessment of ITR-driven luciferase activity compared to a dual luciferase plasmid utilizing strong viral promoters ITR-Luci plasmid contains promoterless Renilla and Firefly luciferase genes cloned into the plasmid backbone adjacent to mITR and ITR sequences, respectively.Psicheck2 is a positive control plasmid utilizing strong viral promoters (SV40 and HSV-TK) to drive Renilla and Firefly luciferase expression, respectively.Luciferase activity was assessed 24 hours after plasmid transfection in HEK293 cells, compared to mock transfected HEK293s.ITR-driven Renilla and Firefly luciferase activity was significantly increased above mock, but less than that achieved by viral promoters.N=3 replicates performed in triplicate.P < 0.05; two-way ANOVA with Dunnett's multiple comparisons test.

Fig S2 .
Fig S2.Locations of mutated sites along the ACTB intron sequences ATG start codons along both halves of the ACTB intron inserted into the proviral backbone.Blue arrows mark the locations where these start codons originally presided before they were mutated from ATG to ATT.Additionally, natural SacII and AseI sites were mutated in the second half of the partial ACTB intron 1 for cloning purposes (marked as black and red arrows).

Fig S3 .
Fig S3.Map of U6.mi405 inserts with C1 or C5 backbones.Relevant features are indicated.Triangles with numbers indicate nucleotide positions.

Table S1 .
Sequences of CTCF DNA-binding sites inserted into ITR-Luci and novel AAV backbone vectors CTCF insulator sequences from Kim et al, "Analysis of the vertebrate insulator protein CTCF binding sites in the human genome", Cell, 2007 Mar 23; 128(6): 1231-1245, Supplementary

Table 7 .
The canonical CTCCC-binding motif is bolded and underlined in each sequence.

Table S3
Next-generation sequencing (NGS) to identify packaged DNA contents of all firstgeneration AAV vectors used in the studyTable included as separate Excel file.AAV vectors carrying the same intended payload (U6.mi405/collagen intron stuffer) were treated with DNase before isolating packaged DNA within indicated AAV capsids.Purified AAV contents were then identified using Illumina HiSeq Table included as separate Excel file.Relevant features are indicated.